The overall goal of our research is to determine the molecular basis of mechanochemical signal transduction in endothelial cells. We have established that temporal gradients in shear stress and steady shear stress represent two distinct mechanical signals that are transduced by two different signaling pathways. The temporal gradient in shear stress has been shown to be a potent stimulator of mitogenic and pro-atherogenic signal and gene expression, and is mediated by the Gq heterotrimeric G protein. In contrast, steady shear stress appears to dose-dependently stimulate anti-atherogenic signals such as prostacyclin and nitric oxide. We hypothesize that mechanoreception of these two mechanical signals occurs at different spatial locations on the cell: temporal gradients in shear stress are perceived at the cell-cell junctions and steady shear is sensed over the luminal membrane. It is the objective of this proposal to elucidate the molecular events that lead to mechanoreception and mechanochemical transduction. Our first two specific aims are to elucidate the molecular associations and sequence of signaling events of Gq and endothelial nitric oxide synthase activation that occur at the cell-cell junctions as endothelial cells are subjected to temporal gradients in shear stress. The third specific aim is to investigate the spatial and temporal pattern of shear stress- induced changes in membrane microviscosity. To accomplish this, we will develop a technique to image membrane microenvironmental changes realtime in endothelial cells subjected to both temporal gradients in shear and steady shear stress. The fourth specific aim will determine if spatial gradients in shear stress do in fact stimulate a proatherogenic phenotype in human endothelial cells. This investigation will test a comprehensive hypothesis on the mechanism of mechanochemical transduction in endothelial cells. If successful, it will provide a fundamental understanding of how the endothelium senses hemodynamic forces in both normal physiology and in vascular disease.